Enzymes constitute a family of proteins involved in catalysing chemical reactions within living organisms. As a result of their importance, there are numerous situations in which it is necessary and/or beneficial to measure enzyme levels, and importantly, enzyme activity.
In particular, increases in enzyme activity have been found to correlate with specific conditions and/or diseases. For example up-regulated protease activity has been associated with many aspects of cancer progression. The measurement of enzyme activity in samples taken from individuals with a particular condition or suspected of having a specific condition or disease may therefore be useful for prognostic or diagnostic purposes.
Within the enzyme family, there are six major categories of enzyme: —oxidoreductases; transferases; hydrolases; lyases; isomerases and ligases. This classification is based upon the type of reactions catalysed by the enzymes within each category. For example, hydrolases typically catalyse the hydrolysis of chemical bonds within their substrates, and include inter alia enzymes such as proteases, peptidases, lipases and nucleases.
Assays have previously been developed for the measurement of enzyme activity; however, these are often associated with problems or limitations. For example, some enzyme detection devices can only be used to measure the activity of enzymes that cleave their substrates. One such device is described in US2006/0003394 involving a kit comprising a substrate-reporter “reactive complex” that is added to a test sample known or suspected of containing the enzyme. The enzyme-reactive complex is applied to a chromatographic medium whereupon immobilisation of the reactive complex occurs at an upstream site. If the substrate is cleaved by any enzyme present within the test sample, the reporter portion of the reactive complex is released and flows to a downstream site where it can be detected separately. Since the detection of enzyme activity is based on measurement of a reporter ‘leaving group’ from the original reactive complex, this device is not at all suitable for measuring the activity of enzymes that modify, rather than cleave, their substrates. For example, transferases catalyse the transfer of a functional group, for example a phosphate group, to their substrates and thus could not be detected using the device described in US2006/0003394.
Different devices have been described that are suitable for detecting the activity of enzymes that modify their substrates. One such device is described in WO2009/024805, which relies on use of a “substrate recognition molecule” (SRM) carrying a detectable label, wherein the SRM specifically binds to the enzyme substrate in either the unmodified or modified state.
Assays such as that described in WO2009/024805, frequently require a very specific detection reagent suitable for discriminating between both modified and unmodified forms of an enzyme substrate and for effective detection. The accuracy and reliability of assay results obtained using such devices can therefore be limited by the availability of suitable reagents.